title: A new model to evaluate Raf signaling in hematopoietic cells.
abstract: The Raf/MEK/ERK pathway is thought to be critical in mediating cell survival and proliferation by cytokine receptors. However, the exact contribution of Raf is complex and not well understood. A better understanding of Raf signaling is important because of the recent observation that B-Raf is frequently mutated in various human cancers. We have generated a new model system that activates Raf directly by linking the extracytoplasmic and transmembrane domains of the erythropoietin receptor (EPOR) with the catalytic domain of Raf (CR3). This synthetic oncogene in which dimerization can be controlled by an exogenous ligand, is fixed at the cellular membrane, while the endogenous Raf is normally activated by binding with Ras. The chimeric receptor EPOR/CR3 was stably expressed in Ba/F3 cells which lack EPO receptors. Although the lines remained dependent on IL-3 for proliferation, EPO treatment reduced the rate of cell death in the absence of IL-3. Also, EPO was synergistic with sub-optimal concentrations of IL-3 in inducing long-term cell proliferation, but did not augment proliferation of cells cultured with full concentrations of IL-3. EPO induced a rapid activation of ERK and also phosphorylation of endogenous Raf. It also induced tyrosine phosphorylation of several cellular proteins. The MEK1 inhibitor PD98059 reduced EPO-induced tyrosine phosphorylation, suggesting these substrates are downstream of MEK kinase. Interestingly, PD98059 also reduced the phosphorylation of endogenous Raf, indicating there is a positive feedback mechanism in Raf activation. We conclude that Raf can be activated by a mechanism that induces clustering at the cell membrane, and that this leads directly to activation of MEK and ERK. This EPOR/CR3 system may serve as a useful model to evaluate the unknown Raf kinase pathway and the effects of signal transduction inhibitors for Raf as a target.
authors.affiliation: Department of Molecular Diagnostics and Therapeutics, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kyoto, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Okuda
mesh_terms: D020134:Catalytic Domain / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics; D049109:Cell Proliferation; D004921:Erythropoietin / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005786:Gene Expression Regulation; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D007377:Interleukin-3 / Q000378:metabolism; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000235:genetics*; D010766:Phosphorylation; D011485:Protein Binding; D017467:Receptors, Erythropoietin / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D048490:raf Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D007377:Interleukin-3; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D048490:raf Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D048369:MAP Kinase Kinase 1